Control of chemical reactions becomes especially challenging when chemical processes have to work in the complexity of biological environments. This is one of the reasons why the ability to design molecules with structure, reactivity and biological activity “switchable” via an externally controlled factor continues to draw significant attention, both from the practical and fundamental points of view. Possible applications of such molecules include design of molecular machines and switches, logic gate mimics, optical sensors, drug delivery systems, etc. Since pH-dependent “switchable” molecules are of particular use for processes that occur in biochemical systems and in the environment, interesting pH-sensitive systems were developed to control such diverse phenomena as strand orientation and exchange in peptide assemblies, charge densities in self-assembled monolayers, encapsulation of guests in supramolecular polymers, phase transitions in stimuli-sensitive polymers for drug delivery, rotaxane switching, properties of luminescent devices based on organic fluorophores and metal complexes, permeability of mesoporous materials, growth of nanomaterials, control of recognition-mediated reactions, and the design of bioreactors. Life itself is a pH sensitive phenomenon, as many biochemical processes work only within a very narrow pH window.
In this study, we provide the first example of a “switchable” molecular system for pH-controlled double stranded DNA-cleavage designed for selective targeting of cancer cells. The more acidic extracellular environment of solid tumors, relative to that of the normal cells, results in a pH gradient that has a dramatic effect on drug uptake in tumor cells and can be explored in the design of tumor-specific DNA cleaving agents. It is known that hyperglycemia (e.g., glucose infusion) and/or certain drugs, e.g., amiloride, nigericin, and hydralyzine, are also able to lower the intracellular pH of cancer cells. For example, administration of amiloride and nigericin at dosages that do not affect the normal cells drops the intracellular pH in a number of tumor cell types from 7.2 to 6.2-6.6.′″ Moreover, hyperglycemia as well as hypoxia lead to an even further acidification to pH as low as 5.5.′
Although research in this direction has been hampered by the scarcity of suitable pH-dependent cytotoxic agents, a number of approaches can be used for the rational design of such molecules. An illustration of how these ideas can be applied to highly potent class of natural enediyne antibiotics, either through isomerization into more reactive functional groups or through unlocking of structural constraints, is given in FIG. 1. In addition, there have been promising reports of acid-labile drugs that either hydrolyze at lower pH to give toxic products or produce free radicals due to accelerated Co—R bond homolysis. An interesting recent finding involves acid-promoted DNA cleavage by natural antibiotic Varacin C. The authors found a two-fold increase in the extent of single stranded (ss) DNA cleavage at pH 5.5 (47% at 5 μM antibiotic loading) compared with that at pH 7 (23%). Unfortunately, this increase only applies to the ss damage which is, unlike the double stranded (ds) damage, usually repairable by the cell chemical machinery.
Another approach to pH-regulated DNA-cleaving agents involves protonation of basic functional groups. Amine functionality is one of the obvious choices and several elegant experimental designs based on the protonation of this functional group have appeared in the literature (FIG. 2). In particular, the groups of Kerwin, Chen, and Kraka & Cremer reported systems where protonation increased the efficiency of radical damage through simultaneous acceleration of the H-abstraction step and deceleration of p-benzyne diradical deactivation through the retro-Bergman ring opening. After detailed computational studies indicated that properly positioned cationic groups decrease the activation barrier for the Bergman cyclization, Basak and coworkers pursued an alternative approach based on a significant acceleration of the cycloaromatization step imposed by a spatially close ammonium moiety.
Unfortunately, simple aliphatic amines are too basic for the change in the protonation state to occur at the pH window necessary for targeting hypoxic cancer cells. As the result, even when protonation has been shown to accelerate the Bergman cyclization of enediynes, the change in reactivity did not occur in the optimal pH-range. Nevertheless, the basicity of nitrogen bases can be controlled in a number of ways and, thus, the above is not insurmountable. For example, anilines are significantly less basic than aliphatic amines and can be fine-tuned through substitution to accept the proton only at the desired pH-range. Amides, suggested by Chen, and aldimines designed by Kraka and Cremer may offer an excellent solution for fine-tuning the nitrogen basicity.